ABSTRACT Glioblastoma (GBM) is the most common and most deadly primary malignant brain tumor. Despite the most advanced treatment with combinations of surgery, radiotherapy and chemotherapy, GBM is associated with a median life expectancy of only ~15 months. Targeted molecular therapies are arguably one of the most promising approaches to achieving more effective future GBM therapies. A major challenge facing such an approach is the simultaneous deregulation of multiple molecules in any given single tumor, as demonstrated by The Cancer Genome Atlas (TCGA) and other published research. Because of this co-deregulation, it is not surprising that molecular monotherapies have failed to achieve significant improvements in GBM clinical outcomes. Several lines of evidence suggest that the simultaneous targeting of multiple deregulated molecules and pathways is required to achieve better therapies. Based on preliminary evidence, we hypothesize that there exist ?master regulatory microRNAs? (miRNAs) that simultaneously regulate multiple deregulated molecules in GBM. The goal of this application is to discover, investigate, and therapeutically exploit such miRNAs. We believe that studying them will provide new information on the mechanisms of gene expression (de)regulation in GBM and that restoring or inhibiting them can be exploited for therapy. We propose three specific aims. In aim 1, we will use a novel screening approach, PAR-CLIP, in combination with smRNA-seq and TCGA gene expression data analysis to uncover global miRNA targets and identify single miRNAs (designated master regulatory miRNAs) that simultaneously target and regulate the largest number of deregulated molecules in GBM. In aim 2, we will investigate the functions and modes of action of these master regulatory miRNAs and validate their expressions and targets in human GBM specimens. In aim 3, we will test miRNAs as novel experimental therapeutic agents or targets in GBM. Thereby, we will develop and use novel potentially clinically applicable local and systemic delivery agents and approaches including brain penetrating nanoparticles (BPN), convection-enhanced delivery (CED) and focused ultrasound with microbubbles (FUS-MB). Successful completion of the proposed studies would establish the first compendium of miRNA targets in GBM, generate new knowledge on the (de)regulation of gene expression by miRNAs and their effects on GBM malignancy, and develop novel technologies for the exploitation of novel master regulatory miRNAs in GBM therapy.